1. Field of the Invention
This invention relates in general to the field of patch-clamp recording and, in particular, to a cartridge system with integrated electrodes suitable for measurements in automated, repeatable, parallel experiments.
2. Description of the Related Art
Conventional voltage clamping techniques used to conduct electrophysiological tests on a membrane assess electrical activity on the membrane by measuring current or voltage changes produced in response to exposure to various test stimuli. Typically, the membrane is pierced with two microelectrodes connected to an amplifier capable of recording current or voltage variations in response to stimuli such as voltage step changes, the application of compounds, or mechanical stimulation.
Similarly, using patch clamping techniques, the membrane potential can be held constant while the current flowing through the membrane is measured to detect ion-channel activity that corresponds to changes in the membrane's conductance. Instead of using sharp microelectrodes to puncture the membrane and penetrate the cell, like in traditional voltage clamping, patch clamping uses a micropipette with a heat-polished tip of about 1 to 5 micron in diameter that is physically sealed to a “patch” on the membrane. The same pipette is used continuously for both current passing and voltage recording. For the most part, patch clamping is used either with a whole-cell or a single-channel mode of operation.
In whole-cell patch clamping, the membrane at the tip of the pipette is ruptured to produce electrical continuity between the electrolyte in the pipette and the interior of the cell. Thus, total membrane current or voltage is measured. In single-channel patch clamping, the integrity of the membrane at the tip of the pipette is preserved. Accordingly, the recorded current is only the current flowing through the patch of the membrane enclosed by the tip of the pipette. Since this area is very small, there is a good chance that only one or a small number of ion channels may be in the membrane patch, and individual ion-channel currents may be recorded.
In both types of patch-clamp techniques, when the tip of the pipette is pressed against the cell membrane, the interior of the pipette is isolated from the extracellular solution by the seal that is formed between the tip of the pipette and the membrane. If the electrical resistance of the seal is sufficiently large, negligible current can leak across the seal and good measurements are obtained. Thus, any leakage of current through the seal is undesirable and the creation of a high-resistance seal (in the order of giga-ohms) is crucial for good results.
The basic design of a patch-clamp recording apparatus includes a chamber filled with an “extracellular” ionic saline solution. Such a chamber could consists simply of a 35 mm Petri dish. A biological membrane or a biological cell that contains ion channels is positioned in the chamber. A patch pipette is fabricated from capillary glass, whereby the tip of the patch has an aperture of one to several microns in diameter. The opposite end of the patch pipette (the pipette “base”) is not modified. The patch pipette is filled with an “intracellular” ionic saline solution and a silver wire coated with silver chloride (the “internal electrode”) is inserted into the patch pipette through the opening in the base. The wire electrode is positioned such that one end of the wire is in contact with the intracellular saline solution while the opposite end is electrically connected to an electrophysiology headstage. A separate “ground electrode,” typically also a silver-chloride coated silver wire, is positioned in the extracellular solution and connected to the electrophysiology amplifier, thus completing the circuit. Electrical current is thus able to flow between the internal electrode and the external electrode via the electrolytes in the intracellular and extracellular saline solutions.
Patch-clamp recording is carried out by positioning the tip of the patch pipette onto the surface of the biological cell (or membrane) and applying suction from the pipette, or other facilitating means, to induce the cell to form a tight seal with the patch pipette. The seal is typically established and monitored by measuring the electrical resistance between the pipette and the cell. An acceptable seal has an electrical resistance on the order of several hundred mega-ohms to several giga-ohms and is often referred to as a giga-seal. Once such a giga-seal has been formed, further suction can be applied to provide whole-cell access to the interior of the cell. This is the most common form of patch-clamp recording. Other variations of patch-clamp recording, such as excised patch, perforated patch, inside-out patch and patch-cramming, are also well known in the art. Once a suitable recording configuration has been obtained, the cell or biological membrane is stimulated and the electrical response is recorded. Common stimuli are also well known in the art.
Planar patch clamping refers to conventional patch clamping wherein multiple cells are recorded at the same time in automatic fashion. Accordingly, planar patch clamping increases the ease, throughput, and reliability of patch-clamp recording. A typical planar patch-clamp apparatus consists of two components with saline-fluid filled chambers separated by a partition with an aperture between each set of chambers. Limiting the description to a single set for ease of discussion, the chambers are typically positioned so that one chamber is above (extracellular chamber) and the other is below (intracellular chamber) the partition. The partition contains a single aperture, approximately one to several microns in diameter, between each set of intracellular and extracellular chambers. Each set of chambers in the planar patch is functionally and structurally substantially the same.
The extracellular chamber is typically filled with extracellular saline solution and a ground electrode is positioned within it, thereby producing a chamber that is functionally equivalent to the extracellular chamber of conventional patch-clamp apparatus. The intracellular chamber is filled with intracellular saline and an internal electrode is similarly placed in it to produce an intracellular chamber that is functionally equivalent to the internal chamber of a patch pipette. The partition between the two chambers is functionally equivalent to the walls of the patch pipette and the aperture in the partition is functionally equivalent to the opening at the tip of a patch pipette. A cell or biological membrane is positioned in the extracellular chamber onto the aperture of the partition. The ground and intracellular electrodes are connected to a ground circuit and a current measurement amplifier, respectively, to complete a circuit.
The electrodes typically consist of a silver wire that has been electrochemically plated with a layer of silver chloride. During use, both the internal and ground electrode are immersed into their respective fluid solutions and the reaction of silver chloride with chloride ions that are typically present in the solutions provides a suitable conductivity for the performance of the electrodes (i.e., low potential drift and little dependence of the electrode potential on electrical current flowing through the electrode). Since the silver-chloride layer slowly dissolves in the saline solutions, the electrodes have a limited life and require periodic replacement or refurbishment (re-coating with silver chloride).
During the process of planar patch-clamp recording, a giga-ohm seal is formed between the biological membrane and the surface of the partition in the region of each aperture. Suction, or other facilitating means, may be used to rupture the membrane in the aperture, thereby providing whole cell access between the interior of the cell and the intracellular chamber through the aperture in the partition. Electrical current flows between the two chambers through the cell and aperture and is monitored via electrophysiology instrumentation.
Planar patch-clamp for high-throughput screening utilizes multiple-chamber components and disposable planar partitions that are typically attached (bonded or reversibly clamped) to the intracellular component or the extracellular component, or both. The entire assembly of the intracellular component, the partition, and the extracellular component is referred to as a patch-clamp cartridge. Each intracellular component contains a plurality of intracellular chambers (for example 16). Accordingly, each extracellular component also contains a plurality of chambers (such as 16). The partition contains the appropriate number of apertures (e.g., 16) to provide a single aperture for each intracellular chamber. Various cartridge configurations may be desirable for different applications, such as with 96, 384, or 1536 intracellular and extracellular chambers.
It is also possible to use a common electrode for one type of component. For example, a common ground electrode can be used for all the chambers in the extracellular component. In some applications, it may be desirable to have a common chamber as well, rather than discrete chambers, in a given component. For example, a common chamber could be used for the intracellular component with a single electrode for that chamber. Such a common chamber could be coupled to multiple extracellular chambers in the extracellular component via a multiple-apertured partition with a single aperture aligned with each extracellular chamber. In such a case, each of the extracellular chambers would require an independent electrode.
FIG. 1 shows a patch-clamp cartridge 10 consisting of an extracellular component 12, a partition 14, and an intracellular component 16. For planar patch clamping, the internal and ground electrodes (not shown in this figure) are positioned in the intracellular chambers 18 and the extracellular chambers 20, respectively. The electrodes are positioned to contact the solutions in the fluid chambers so that ion current measurement can take place. To be performed automatically, such positioning of the electrodes requires two separate mechanisms, as each electrode is typically positioned on opposite sides of the partition.
This invention is directed at improving the design of patch-clamp cartridges to provide more efficient fluid delivery and electrode operation. It is understood that the concepts described herein are applicable to all types of patch-clamp cartridges used in the art, including those with asymmetrically chambered intracellular and extracellular components.